Changes in the genome of African swine fever virus (Asfarviridae: Asfivirus: African swine fever virus) associated with adaptation to reproduction in continuous cell culture

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Introduction

African swine fever virus (ASFV) is a large (175–215 nm), double-stranded DNA virus in the Asfarviridae family. It is the causative agent of African swine fever (ASF), a haemorrhagic disease with high mortality rates in domestic pigs.

To study the foundations of the pathogenicity and immunogenicity of the ASFV, it is necessary to carry out a comparative analysis of the biological properties of the virus and the structure of its genes. ASFV is able to replicate in porcine primary cell cultures (CC), such as porcine leukocytes CC, alveolar macrophages, etc. However, these cultures are difficult to standardize because their properties differ depending on the characteristics of the animal used, which makes it much more difficult to obtain a genetically uniform sample of virus suitable for analysis. To address this problem, various ASFV isolates have been adapted to grow in continuous cell culture, ex. Georgia 2007/1 and BA71 in Vero cell culture. As a result of this adaptation, ASFV-G/V strain and the BA71V strain were obtained [1][2]. Although, both these strains were reported to be avirulent following adaptation, only the genome of BA71V was sequenced and analyzed [2].

In the process of adaptation to growth in continuous cell cultures, the virulence of the ASFV decreased [3]. The purpose of this work was a comparative analysis of the nucleotide sequence obtained as a result of the whole genome sequencing of two strains of the ASFV obtained after 30 and 50 passages in the CV-1 cell culture, with the genome of the original isolate.

Previous studies indicated a decrease in virus virulence of isolate ASFV/ARRIAH/CV-1/30, yet the genome of this strain following adaptation was not well characterized [3]. In this study, we investigate how ASFV adaptation to growth in continuous cell culture might influence the virus’s genome, and identify the resulting mutations.

Material and methods

Strains ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 were propagated in CV-1 cell culture, as described previously [4]. Genomic DNA (gDNA) of the ASFVs were extracted using the previously described phenol-chloroform method and the resulting gDNA pellets diluted in nuclease-free water [5]. A sequencing library was constructed using the «Nextera XT DNA library preparation kit» (Illumina, USA) and Next Generation Sequencing (NGS) was performed using a «MiSeq reagent kit version 2» with 2 × 250-bp paired-end sequencing on a MiSeq benchtop sequencer (Illumina, USA). In order to assemble each of the genomes, reads were mapped to the reference genome (FR682468.1_ASFV/Georgia 2007/1) and a consensus sequence was generated for each of the isolates (CLC Genomics Workbench v.9.5.2 (QIAGEN, Aarhus; www.clcbio.com). The original set of reads were subsequently mapped to the newly assembled virus genome, with an average coverage depth of 45× and an average read length of 250 nucleotides (nt). Open reading frames (ORFs) were predicted using GATU software. The complete genome sequences of both high passage strains were deposited in GenBank with accession numbers MW528217 and MW528218.

The complete genome sequences of previously characterized ASFV isolate Odintosovo 02/14 (KP843857.1) and the genotype II reference, ASFV/Georgia 2007/1 (FR682468.1), were included in subsequent genetic analysis. The sequences of two reference and the two cell culture adapted strains were used to generate an alignment, which in turn was used to detect single nucleotide polymorphisms (SNPs). Both the construction of the alignment and SNPs detections were performed using CLC Genomics Workbench v.9.

Results

Since the biological and genetic characteristics of the ASFV isolate Odintsovo 02/14 has been studied, the isolate was used during progressive adaption of the virus to continuous cell culture using fibroblast-like pseudodiploid cells from the African green monkey (Cercopithecus aethiops) (CV-1) [6]. The complete genomes of cell culture adapted variants from passage 30 and 50 (referred to as: ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV1/50 respectively) were determined.

The complete genomes of the analyzed viruses differ in length with ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 consisting of 186,529 bp and 186,525 bp respectively, while in comparison the reference strain Georgia 2007/1 and parental isolate Odintsovo 02/14 was 189,344 bp and 189,122 bp respectively. This indicates a large deletion within the genomes of the highly passaged strains. Total 78 SNPs were identified between the parental Odintsovo 02/14 and the two high passaged strains. These could be further classified as 13 SNPs within the intergenic regions and 5 associated with homopolymer repeat sequences. The SNPs within open reading frames were characterized as 8 synonymous SNPs and 52 non-synonymous SNPs. All 60 synonymous and non-synonymous SNPs are listed in Table 1.

Predicted proteins with conservative non-synonymous single nucleotide polymorphisms and synonymous in light grey. The amino acid predicted for each isolate and strain is listed as well as its position. Amino acid exchanges marked with dark grey indicate positions where either ASFV/ARRIAH/CV-1/30 or Odintsovo 02/14 were affected.

The majority (n = 36) of the non-synonymous SNPs are conservative amino acid exchanges, while 14 resulted in an exchange of a charged amino acid with either an amino acid with a different or no charge. Two (n = 2) of the non-synonymous SNPs resulted in early termination of the predicted protein (Table 1 and Table 2). The predicted proteins affected by the early termination as well as the non-conservative amino acid exchanges are listed in Table 2.

The presented results clearly demonstrate that the majority of amino acid exchanges occurred in the genome of the adapted ASFV/ARRIAH/CV-1/50 and/or ASFV/ARRIAH/CV-1/30 variants, with the exception of the mutations highlghted in gray, which were unique to the genome of isolate Odintsovo 02/14.

Discussion

Under certain conditions, ASFV, a double-stranded DNA virus, may not have a high level of genome variability, but since no effective vaccine have been developed till the moment against African swine fever, it is of high importance to understand and study the relation between certain biological properties and changes in the virus genome.

Since it was previously described how adaptation of the ASFV to growth in a continuous cell culture leads to significant changes in its genome and a decrease in virulence [3][7], complete genome sequencing and analysis is the most informative approach to understand the effect of these mutations on the pathogenesis of the virus.

In the presented study, it is clearly demonstrated that adaptation of the ASFV to growth in CV-1 continuous cell culture leads to the appearance of a large deletion in the 3’ variable region of the genome. Comparative analysis of the genome of the original isolate and the adapted variants also revealed 78 oligonucleotide polymorphisms that influenced or did not affect the predicted amino acid sequence of the encoded protein.

Only 2 oligonucleotide substitutions in the MGF_110_1L and B354L genes led to the formation of stop codons, which makes it possible to exclude the possibility of a complete synthesis of proteins encoded by these genes when pigs are infected with ASFV/ARRIAH/CV1/50 strain.

Conclusion

The most significant modification in the genomes of ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 was a large-size (2947 bp) deletion in the right variable region of adapted viruses, between 181,980–184,929 bp. A similar deletion was described in the genome of another cell culture adapted strain, BA71V, where the same seven predicted proteins were affected (Table 2) [2]. The importance of each of the described mutations on cell adaptation, virus growth and virulence requires additional investigations. 

About the authors

A. Mazloum

FSBI «Federal Centre for Animal Health» («ARRIAH»)

Author for correspondence.
Email: ali.mazloum6@gmail.com
ORCID iD: 0000-0002-5982-8393

Mazloum Ali, Ph.D. (Biol.), Leading Veterinarian.

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia

Россия

A. S. Igolkin

FSBI «Federal Centre for Animal Health» («ARRIAH»)

Email: fake@neicon.ru
ORCID iD: 0000-0002-5438-8026

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia

Россия

N. G. Zinyakov

FSBI «Federal Centre for Animal Health» («ARRIAH»)

Email: fake@neicon.ru
ORCID iD: 0000-0002-3015-5594

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia

Россия

A. van Schalkwyk

Onderstepoort Veterinary Institute

Email: fake@neicon.ru
ORCID iD: 0000-0003-4761-8767

100 Old Soutpan road, Onderstepoort 0110

ЮАР

N. N. Vlasova

Onderstepoort Veterinary Institute

Email: fake@neicon.ru
ORCID iD: 0000-0001-8707-7710

100 Old Soutpan road, Onderstepoort 0110

Россия

References

Supplementary files